This invention relates to the cloning, sequencing and expression of the structural genes of western equine encephalitis (WEE) virus strain 71V-1658 and the development and use of the DNA-based vaccine against WEE.
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The alphaviruses are a group of about 27 enveloped viruses with a positive sense, nonsegmented single-stranded RNA genome (Calisher et al., 1980; Strauss and Strauss, 1988). The alphavirus disclosed in this invention, western equine encephalitis virus (WEE), is a member of the WEE antigenic complex and is serologically related to the Sindbis (SIN), Highlands J (HJ), Fort Morgan, Buggy Creek, and Aura viruses (Calisher and Karabatsos, 1988; Calisher et al., 1988). WEE is endemic in western North America and strains/varieties have been isolated from Argentina (AG80-646), Brazil (BeAr 102091) and the former Soviet Union (Y62-33) (Johnson and Peters, 1996; Weaver et al., 1997). In nature, WEE is transmitted from its amplifying hosts or reservoir in wild birds, to man and horses, by mosquitoes (Culex tarsalis being the principal vector). While the endemic cycle has resulted in only a limited number of human infections in recent years, in the past, major epidemics of WEE have been recorded. The most extensive epidemic, including 3,336 recognized human cases and 300,000 cases of encephalitis in horses and mules, occurred in the western United States and Canada in 1941 (Reisen and Monath, 1988; Johnson and Peters, 1996).
All alphaviruses share a number of structural, sequence, and functional similarities, including a genome with two polyprotein gene clusters (reviewed in Strauss and Strauss, 1994; Schlesinger and Schlesinger 1996). The genomic organization of these viruses is conserved (see FIG. 1), with the nonstructural proteins translated directly from the 5xe2x80x2 two-thirds of the genomic RNA. A subgenomic positive-stranded RNA (the 26S RNA), is identical to the 3xe2x80x2 one-third of the genomic RNA and serves as the translational template for the structural proteins (capsid, E3, E2,6K and E1).
The nonstructural proteins (nsP1, nsP2, nsP3 and nsP4) are also synthesized as a polyprotein and processed into the four nsPs by a nsP2 protease. Two versions of the nonstructural polyprotein are synthesized in alphavirus-infected cells, due to frequent readthrough of an opal codon between the nsP3 and nsP4 genes in several alphaviruses (Strauss et al., 1983). The nsPs function in a complex with host factors to replicate the genome and transcribe the subgenomic mRNA. Alphaviruses have characteristic conserved sequences at the extreme 5xe2x80x2 and 3xe2x80x2 domains and the intergenic region (Ou et al., 1982, 1983; Pfeffer et al., 1998). These conserved domains are required for viral growth and replication and are believed to be important in promotion of protein synthesis and the initiation of RNA-dependent RNA polymerase activity.
The relationship of different WEE isolates to each other has been demonstrated using neutralization tests (Calisher et al., 1988). Additionally, several strains of WEE were typed by oligonucleotide fingerprinting, and found to have greater than 90% nt homology (Trent and Grant, 1980). The N-terminal sequences of the nucleocapsid, and the E1 and E2 glycoproteins have been determined by Edman degradation, and the E1 and E2 proteins were found to have 82% and 71% homology, respectively, to SIN (Bell et al, 1983). Hahn et al. (1988) sequenced the 26S region of WEE strain BFS1703. They proposed that WEE originated as a hybrid virus, formed by recombination of an EEE and a Sindbis-like virus, most likely during a co-infection event. They suggested that two crossover events occurred, one within the E3 gene, the other within the 3xe2x80x2 nontranslated terminal region (NTR), resulting in a virus whose nonstructural domain, intragenic region, and capsid protein are similar to EEE, with envelope proteins showing homology to SIN.
Weaver et al. (1993) sequenced part of the nonstructural domain (nsP2 and nsP3 genes) of strain 5614, demonstrating this area also shows homology to EEE. Short regions within the nsP4 gene and the E1 protein/3xe2x80x2 NTR have been determined for many WEE strains, allowing a preliminary assessment of the nucleic acid phylogenetic relationships within the WEE antigenic complex (Weaver et al., 1997). Serological studies (Calisher et al., 1988) and preliminary sequence determination (Cilnis et al., 1996; Weaver et al., 1997) of the HJ genome suggests this is another closely related virus, and most likely a descendant of the same recombinant viral ancestor as modem WEE.
A highly conserved region of the alphavirus nsP1 gene has been identified, and proved suitable for use in a polymerase chain reaction (PCR)-based genetic assay for alphaviruses, including WEE (Pfeffer et al., 1997). Phylogenetic analysis of this PCR fragment yielded similar results to those obtained by Weaver et al., (1997) for a PCR fragment in the nsP4 gene.
In terms of therapy or prophylaxis, there are very limited possibilities. An inactivated vaccine to WEE is under investigational new drug (IND) status. The vaccine uses formalin-inactivation of cell culture supernatants from WEE-infected tissue culture. It requires a minimum of 3 doses, yearly monitoring of antibody titer and possible boosters. Its effectiveness in the protection against an aerosol challenge of WEE has yet to be established. A WEE live attenuated vaccine based on an infectious clone is under development (J. Smith, personnel communication). The area of DNA immunization is relatively new, and has been reviewed in Hassett and Whitton, 1996; Donnelly et al, 1997. Similar to live, attenuated vaccines, DNA vaccines are known to stimulate both humoral and cellular immune responses (Pardoll and Backering, 1997; McCuskie and Davies, 1999). Much of the focus has been on methods to deliver and efficiently express the cloned products. Intramuscular administration of DNA has been one of the original methods used (Wolff et al, 1990). A second method uses ballistic delivery of DNA coated gold particles, using high pressure helium gas to propel the particles into the epidermis and dermis of animals (Prayaga et al, 1995, reviewed by Robinson et al, 1995).
The Applicant identified a number of related areas of research, including the development of subunit vaccines to WEE. In the present invention, the Applicant disclosed the cloning, sequencing and expression of the structural genes of a WEE virus (strain 71V-1658), as described in Netolitzky et al., (2000) xe2x80x9cComplete genomic RNA sequence of western equine encephalitis virus and expression of the structural genes.xe2x80x9d Journal of General Virology 81, 151-159, which is herein incorporated by reference. The DNA construct (pCXH-3), and a second construct (pVHX-6) were used in DNA immunization studies in a mouse model for protection against intranasal administered WEE.
The present invention is directed to the development of a DNA-subunit vaccine to the WEE virus and its use against such virus. More specifically, DNA to structural components of the WEE virus are expressed and used as the subunit vaccine.
The present invention provides for the complete nucleotide sequence of WEE strain 71V-1658. Two novel cDNA clones, pCXH-3 and pVHX-6 are also disclosed as effective vectors for gene expression.
The present invention also provides the complete nucleotide sequence for the structural gene pcDWXH-7.
It further provides for a process for preparing a recombinant DNA vaccine against WEE virus, comprising cloning and sequencing of 26S region of a WEE virus strain 71V-1658 under conditions suitable to effect in vitro transcription and translation of the functional recombinant DNA expression vector pCXH-3 and pVHX-6.